Although it is known that the mammalian epidermis is maintained by self-renewal of stem cells, the underlying mechanisms, and the changes that occur in skin cancer, are not well-defined. A more thorough understanding of stem cell proliferation and differentiation will provide potential targets for skin cancer prevention. One of the most widely used models for the study of skin carcinogenesis is the mouse model of initiation and promotion. The initiating event occurs when exposure to a carcinogen induces genetic changes in epidermal stem cells, which are thought to be located in the bulge region of the hair follicle. Initiation causes activation of the Ha-ras oncogene and chronic up-regulation of the Raf/MEK/ERK pathway. Our previous work demonstrates that the polyamine metabolizing enzyme ornithine decarboxylase (ODC) is downstream of MEK, and induction of ODC activity is necessary for skin tumor development. We have also shown that antizyme (AZ), which binds to ODC and targets it for degradation, is a potent tumor suppressor in skin carcinogenesis. The central hypothesis of the proposed work is that the tumor suppressor effects of AZ are associated with decreased proliferation of epidermal stem cells and induction of keratinocyte differentiation. The proposed experiments use mice with expression of AZ in the skin, along with either MEK or cyclin D1 (CD1), which like ODC is a downstream target of MEK. Expression of each transgene is driven by either the Keratin 5 or Keratin 14 promoter, thus directing expression primarily to the hair follicle bulge and modifying important signal transduction pathways in the population of cells crucial to stem cell expansion, keratinocyte differentiation and tumorigenesis. Preliminary studies with mice expressing both AZ and CD1 have led to the hypothesis that reduced ODC activity resulting from targeted AZ expression restricts cell growth in epidermal stem cells and inhibits tumorigenesis at least in part by suppressing CD1 activity. Aim 1 will measure stem cell expansion in bulge cells of MEK, MEK/AZ, CD1 and CD1/AZ mice using BrdU labeling and expression of a6-integrin and CD34, both markers of epidermal stem cells. We will directly address whether CD1 is controlled by ODC in bulge cells by isolating these cells from several transgenic lines using FACS sorting and monitoring CD1 expression. Aim 2 will use organotypic raft cultures of human keratinocytes expressing MEK, CD1 and/or AZ. This three-dimensional model system will allow us to address changes in tissue organization and expression of differentiation markers in response to transgene expression. The possibility that AZ is acting independently of ODC will be addressed by comparing results in AZ-expressing cells to those in cells expressing siRNA targeted to the ODC protein, thus knocking down ODC without changing AZ expression. To extend these studies to an in vivo model, human keratinocytes expressing our genes of interest will be grafted onto athymic mice, and changes in epidermal proliferation and differentiation will be measured. Information obtained from the proposed work will lead to future studies that address the broader question of what pathways are altered in follicular stem cells upon ODC downregulation.